Tetracyclines are a large group of drugs with a common basic structure consisting of four linearly fused six-membered rings. Chlortetracycline isolated from Streptomyces aureofaciens was introduced in 1948 and oxytetracycline, derived from Streptomyces rinosus, was introduced in 1950 (Projan et al., 2006. SIM News 55, 52-60). Tetracycline and 6-demethyl-7-chlortetracycline (demethylchlortetracycline), both produced by Streptomyces aureofaciens, are two additional tetracycline compounds produced by fermentation process. A number of semi-synthetic tetracyclines generated by chemical modification of tetracycline or demeclocycline and with improved pharmacological properties, have been generated over the years such as methacycline, doxycycline and minocycline. Recently, a novel semisynthetic analogue, tigecyclin, derived from minocycline has been licensed for treatment of bacterial infections (Chopra et al., Roberts M. 2001. Microbiol Mol Biol Rev; 65:232-260). Tetracyclines were the first broad-spectrum antibiotics. They are effective against a variety of microorganisms and are thus often used indiscriminately. Tetracyclines bind reversibly to the 30S subunit of the bacterial ribosome in a position that blocks the binding of the aminoacyl-tRNA to the acceptor site on the mRNA-ribosome complex. They are bacteriostatic for many gram positive and gram negative bacteria, including some anaerobes, for rickettsiae, chlamidiae, mycoplasmas and L-forms, and for some protozoan parasites. The widespread use of tetracyclines has led to the emergence of resistance even among highly susceptible species such as pneumococci and group A streptococci. For this reason a novel antibacterial is needed; tetracyclines, as relatively safe antibiotics, still represent potentially useful candidates for antibacterial drug discovery programmes. The tetracycline analogue doxycycline has been used for decades for the inhibition of the malaria-causing Plasmodium falciparum. Novel tetracycline (TC) analogues have been developed in the past (Projan et al., 2006. SIM News 55, 52-60). A small group of TC analogues, of which the primary target is not a bacterial ribosome, such as chelocardin and 6-thiatetracycline, have been isolated or synthesised (Chopra I. 2004. Antimicrob Agents Chemother; 38(4):637-40).
This small group of TC analogues are bactericidal, rather than bacteriostatic. Their mode of action is clearly not oriented towards a bacterial ribosome. It is believed that the primary target of these small group of tetracycline analogues, such as chelocardin, is the bacterial cytoplasmic membrane, hence the activity of these compounds against tetracycline resistant strains. This has been suggested in the study by Olivia et al. (1992, Antimicrob Agents Chemother. 36(5): 913-919), in which the activities of these tetracycline analogues were examined against E. coli and Staphylococcus aureus strains containing determinants for efflux Tet(B) and Tet(K) or ribosomal protection Tet(M). Chopra et al. (2001, Curr Opin Pharmacol. 1(5):464-9) have demonstrated that Tet(B) and Tet(M) determinants in E. coli or Staphylococcus aureus offer little or no protection against the tetracycline analogues chelocardin and 6-thiatetracycline, thus representing an interesting antibacterial activity. Unfortunately, the clinical trials conducted with one of these atypical TCs (6-thiatetracycline) have revealed adverse side-effects. LD50 of both, 6-thiatetracycline and chelocardin, obtained in acute toxicity studies in mice, are considerably lower that those obtained by classical TCs, possibly reflecting the membrane-disruptive properties of atypical TCs. In view of this potential mode of action it is not surprising that the atypical TCs exhibit activity against TC-resistant strains. Therefore, the use of atypical TCs (such as chelocardin) can not be considered because of their potential for causing side effects. Selected tetracycline analogues have also displayed potent antifungal activity. Several chemically modified tetracycline analogues (CMTs), which were chemically modified to eliminate their antibacterial efficacy, such as CMT3, were found to have potent antifungal properties (Liu et al., 2002, Antimicrob agents chemother 46, 1447-1454).
Tetracycline analogues, including the medically important tetracycline analogues, show other, non-antibacterial pharmacological properties, therefore showing useful activity for the treatment of chronic neurodegenerative diseases (Parkinson's, Huntington's) and autoimmune condition multiple sclerosis (Domercq and Matute, 2004, Trends Pharmacol Sci. 2004. 25(12):609-12, Brundula et al., 2002, Brain 125: 1297-308). In addition, some of the TCs inhibit the activity of matrix metalloproteinases (MMPs), which play an important role in the development of atherosclerosis, rheumatoid arthritis, osteoporosis, tumour invasion and metastasis (cancer development/progression) (Fife et al., 2000, Cancer Lett. 29; 153(1-2):75-8). Pathologies that are responsive to tetracycline compounds include inflammatory process-associated states. The term “inflammatory process-associated state” includes states involving inflammation or inflammatory factors such as MMPs.
Some of these MMPs break down fibrillar collagens and are known as collagenases (e.g. MMP-1, MMP-8 and MMP-13), some can affect basement membrane collagen (collagen IV) and are known as gelatinases (MMP-2 and MMP-9). Tetracycline analogues can inhibit both collagenases and gelatinases (Peterson J. T. 2004, Heart Fail Rev., 9, 63-79). MMPs-degrading enzymes (e.g. MMP-8, MMP-9), present in the intracellular matrix of tissue facilitate angiogenesis by allowing new blood vessels to penetrate into the matrix. Currently only Periostat® (CollaGenex Pharmaceuticals Inc.), also known as doxycycline, is approved for treatment of adult peridontitis, as an MMP inhibitor. The anti-angiogenic effect of tetracyclines may have therapeutic implications in inflammatory processes accompanied by new blood vessel formation, as it is the case in some stages of autoimmune disorders and cancer invasion. Metastat (Col-3), for example, has demonstrated good results in the treatment of Karposi's sarcoma (Phase II, Dezube et al., 2006, J Clin Oncol. 24(9):1389-94). TCs can also influence bone metabolism. Prophylactic administration of doxycycline reduces the severity of canine osteoarthritis in the dog anterior cruciate model (Yu et al., 1992. Arthritis Rheum. 1992 October; 35(10):1150-9). In a recent experiment it was demonstrated that minocycline, by stimulating new bone formation, prevents the decrease in mineral density (osteoporosis) observed in ovariestomized old rats (Wiliams et al., 1998. Adv Dent Res. 1998 November; 12(2):71-5), suggesting the potential use of TCs in the treatment of osteoporosis. Nevertheless, tetracyclines have been shown to demonstrate anti-inflammatory properties, antiviral properties and immunosuppressive properties. The tetracycline analogue minocycline, for example, is considered as a safe effective treatment for patients with mild to moderate rheumatoid arthritis. Tilley et al. 1995 (Ann Intern Med., 122, 2. 1995, 81-89.) carried out a clinical trial in which 109 patients on minocycline were compared to 110 patients on placebo. There was a significant improvement in joint swelling in the treated patients versus the placebo group and also improvement in joint tenderness, with no serious toxicity.
To date, all clinically useful TC antibiotics are either natural products, semisynthetic analogues, or chemically modified molecules, composed of four rings, designated A, B, C, and D (FIG. 1). The recently established crystal structure of tetracycline (TC)-bound 30s subunit (Brodersen et al., 2000, Cell, 103:1143-54.) has revealed that the side of the four-member ring structure of TC molecule, including carbons C1 to C3 and C10 to C12 (“south” and “east” side) interact significantly with the ribosome. Most semisynthetic tetracycline analogues with superior antibacterial activity, such as doxycycline, minocycline and the latest derivative tigecycline, have been modified at the “north-west” side of the tetracycline structure, covering carbons C4 to C9, which is in line with the structure-activity (SAR) results (Brodersen et al., 2000, Cell, 103:1143-54). The structure of chelocardin, in particular, differs from existing biosynthetically-derived natural tetracyclines, thus allowing novel chemistry to be carried out on the tetracycline backbone of chelocardin or modified matrices generated by biosynthetic-engineering approaches, which is the main scope of the invention. Combined synthetic and biosynthetic complementary strategies for novel TC compounds can be applied. The four ring naphtacene nucleus structure of chelocardin and a complex series of oxygen functional groups on the “south” side of the molecule fulfill the minimal structural requirements for bioactivity against both bacterial and mammalian targets. However, the structure of chelocardin is extremely non-polar, compared to other biosynthetic TC derivatives, which is a consequence of the lack of hydroxyl groups at positions C5 and C6, and the replacement of the amino group of the amide moiety at the position C2 with acyl. An addition, the methyl-group at the position C9 further enhances the non-polar properties of chelocardin at the same time altering/broadening the spectrum of biological targets, not only limited to bacterial cells. On the other hand, the free amino-group, not found “unprotected” in other natural tetracycline analogues introduces a degree of polarity. At the same time, it is one of the most useful functional groups that can be readily derivatized by a chemical synthesis approach, introducing changes in solubility, lipophilicity and new binding affinities into the molecule.
In the past, extensive data on the structure-activity relationship of TCs have been generated, showing that the molecular structure and functionality of different TCs allows them to be “chemically promiscuous” and interact with many macromolecules, hence exerting a broad spectrum of pharmacological effects. The present invention is related to the generation of novel TC analogues based on chelocardin itself and/or chelocardin analogues generated by methods of biosynthetic engineering, biotransformation and/or semisynthetic approaches. A more detailed description of the present invention is provided herein below.